Project Summary Infections with Mycobacterium tuberculosis (Mtb) result in over 1.5 million deaths annually. Unfortunately, there are no effective vaccines against pulmonary disease and current treatment regimens extend to at least six months. There is an urgent need to develop new approaches to treatment, such as host-directed therapies, that shorten treatment time and improve disease prognosis. To effectively develop new host-directed therapies, we must understand how protective pathways function mechanistically and determine how they interact with other immune networks. Using targeted genetic interaction studies, we recently discovered a strong synthetic lethal interaction between the NADPH phagocyte oxidase (Phox) and Caspase1/11 (Casp1/11) during Mtb infection. Loss of either locus individually results in minimal survival defects yet combining deletions resulted in rapid disease progression within 4 weeks post-infection. These infections were characterized by increased Mtb in the lungs and dysregulated inflammatory signals including IL1a and IL10. Here the failed host response in Phox/Casp1/11 animals will be dissected. The susceptibility of Phox/Casp1/11 animals might be due to loss of resistance mechanisms that directly control pathogen replication and/or tolerance mechanisms that control overall host health during infection. We hypothesize that loss of Phox and Caspase1/11 together results in dysregulated immune defense cascades leading to both failed resistance and tolerance during Mtb infection. We have developed models that allow the effects of tolerance and resistance to be quantified. In Aim1 defects in the antimicrobial resistance of Phox/Casp1/11 animals will be measured using ex vivo and in vivo models designed to report directly on bacterial growth and fitness independently of inflammatory differences. In Aim 2 tolerance responses in Phox/Casp1/11 animals will be examined using an in vivo model that normalizes Mtb levels between animals and directly quantifies how dysregulated inflammation contributes to susceptibility. Determining the contribution of resistance and tolerance to the susceptibility of Phox/Casp1/11 animals is a critical step to define the immune networks modulated by Phox and Casp1/11 and examine their potential as host-directed therapy targets.